The development of transgenic crops requires methods for the stable insertion of recombinant DNA, a process typically referred to as transformation. Transformed plant cells are generated by the use of a variety of methods to insert foreign DNA into plant cells. These methods further employ the use of genetic constructs which contain genes encoding resistance to chemical selection agents such as antibiotics (e.g. kanamycin or hygromycin) or herbicides. Plant cells expressing selectable markers are preferentially obtained by culturing a population of plant cells subjected to a transformation process in the presence of media which contains, in addition to the selective agent, specific combinations of phytohormones and nutrients to allow cells to grow and regenerate to intact plants. Thus, the process of producing a transformed cell requires a means to physically insert foreign DNA as well as a means to select for those cells which have incorporated the DNA in an expressible and stable form.
Following the stable insertion and expression of the foreign DNA, regeneration of cells containing the foreign DNA leads to the recovery of whole plants, usually following shoot-, embryo- or organo-genesis. The regenerated plant tissue is induced, by a number of different means, typically by the alteration of tissue culture conditions, more typically by manipulation of phytohormones, to form a whole plant capable of forming seed and/or pollen, which can transfer the foreign DNA to subsequent generations. In some plant species, a major limitation is efficient selection and regeneration of plant cells following transfer of DNA, as opposed to the actual transfer of DNA into the cell. Thus, practical limitations on the use of standard transformation methods generally relate to the efficiency of selection and identification of transformed plant cells, not necessarily the transfer of DNA into the plant cell. Another step in the process that is often problematic is the regeneration of transformed cells into whole plants in the presence of a selective agent.
One potential problem related to the selection of plant cells is the effect of the selective agent on non-transformed cells, i.e. plant cells that do not contain the selectable marker. Often the selection process kills the majority of non-transformed cells, a process that, for a variety of physiological reasons, often reduces the efficiency or frequency of regeneration of transformed cells. Thus, the selective pressure needed to identify the cells containing the foreign DNA often proves to be an inhibitory factor to the efficient regeneration of plant cells.
Thus, methods that allow for the selection of plant cells without a direct lethal effect on the non-transformed cells may provide a means to reliably recover transformed plant cells. In addition, regeneration may prove to be more efficient, especially in those plant species, varieties or cells that are difficult to transform by means conventionally practiced. This problem is a major limitation for the routine and economical production of transformed plants.
It is believed that transformation of plant tissue results in only a small percentage of the total number of cells subjected to the transformation process actually incorporating the DNA into their nuclear DNA. Upon application of the selective agent, most plant cells die and may release substances (such as ethylene or senescence related compounds) that affect other nearby cells, including those containing the selectable marker gene and thus prevent efficient regeneration of these transformed cells. It appears that the death of the non-transformed cells may release substances inhibitory to the survival of the transformed cells. Accordingly a primary limitation for efficient production of transformed plants is the recovery of transformed plant cells and subsequent regeneration to whole plants. Methods which permit the rapid and efficient recovery of plant cells containing foreign DNA under conditions where the selection process does not lead to the death of non-transformed cells, (with subsequent inhibition of regeneration) would provide a convenient and efficient means to recover plants, particularly in those plant species where transformation has proven to be costly and time-consuming. Many plant species are capable of being transformed by introduced DNA, however the efficiency of the regeneration process is exceedingly low and prohibits the development of transgenic crops. Furthermore, the significant variation in tissue culture response or regeneration potential in tissue culture found within species, subspecies or varieties within a specific crop genus severely restricts the effective range of current transformation processes.
Brassica species are of special interest. Transformation of members of the Cruciferae family by Agrobacterium and other methods has been reported. A significant body of scientific work has accumulated that describes various methodologies, techniques and studies related to the generation of transgenic Brassica plants that contain foreign DNA of commercial interest and utility. A significant effort to achieve this was made through the early 1980's and still is being made. The technology has been advanced to the point where transformation is now routine for certain narrow genotypes of a species within the Brassicacea, however, it remains difficult within other species in the same family. In addition there is a strong influence of genotype on transformation success within those Brassicas that have been successfully transformed and technology that is more widely applicable to even the “recalcitrant” genotypes would be of great value to the industry. While there are many transgenic Brassica undergoing field evaluation, analysis of the field trial data clearly indicates that the majority (>95%) of the transgenic Brassica are B. napus types, much of the material has been derived from a narrow range of germplasm within B. napus. Part of the reason for this has been the difficulty of routinely obtaining transformed B. rapa and oleracea types as well as the fact that the development of commercial transgenic plants is still relatively recent.
However, many of the reports that relate specifically to Brassica transformation have detailed the difficulty in routinely obtaining transformed Brassica species by Agrobacterium mediated transformation. Many of the reports have shown success with one or two particular varieties, but there is no teaching of detailed methods that are generally applicable to all species within the Brassica genus. Although many manipulations of culture conditions can be employed, some varieties have proven to be extremely difficult to transform by previously reported methods.
Many of the initial Brassica transformation studies were carried out with B. napus cv Westar (Radke et al, Theor. Appl. Genet. 75:685–694, 1988; Moloney et al., Plant Cell Reports 8:238–242, 1989; Moloney et al., 1989, U.S. Pat. No. 5,188,958; Moloney et al., 1989, U.S. Pat. No. 5,463,174). Westar was a convenient choice since it responded to tissue culture and transformation protocols described in the references cited above and allowed recovery of transgenic plants. However; many Brassica transformation studies conducted using the described methods, or variations thereof, have produced results that are highly variable and are dependent upon the innate response of the specific plant materials to the transformation protocol. As an example, the transformation frequencies that have been achieved for Brassica napus are sometimes variable and very low (Fry et al., Plant Cell Reports 6:321–325, 1987; Mehra-Palta et al., In Proc 8th Int. Rapeseed Congress, Saskatoon, Saskatchewan, 1991; Swanson and Erickson, Theor. Appl. Genet. 78:831–835, 1989). Variable and often low transformation frequencies have also been observed with other Brassica species, such as B. oleracea (Christie and Earle, In Proc 5th Crucifer Genetics Workshop, Davis, pp 46–47, 1989; Metz et al., Plant Cell Reports 15: 287–292, 1995; Eimert and Siegemund, Plant Molec. Biol. 19:485–490, 1992; DeBlock et al., Plant Physiol. 91:694–701, 1989; Berthomieu and Jouanin, Plant Cell Reports 11:334–338; Toriyama et al, Theor. Appl. Genet. 81:769–776, 1991); B. rapa (Radke et al., Plant Cell Reports 11:499–505, 1992; Mukhopadhyay et al, Plant Cell Reports 11:506–513, 1992); B. juncea (Barfield and Pua, Plant Cell Reports 10:308–314, 1991; Deepak et al., Plant Cell Reports 12:462–467, 1993; Pua and Lee, Planta 196:69–76, 1995); B. nigra (Gupta et al, Plant Cell Reports 12:418–421, 1993); and B. carinata (Narasimhulu et al., Plant Cell Reports 11:359–362, 1992; Babic, M.Sc. Thesis, Univ of Saskatchewan, 1994).
The many Brassica species, varieties and cultivars represent a very diverse group with radically different morphologies and physiological characteristics. In addition to oilseed Brassica, the vegetable Brassicas represent a crop with significant economic value. This value could be enhanced by the addition of certain novel traits such as disease and insect resistance, male sterility systems for hybrid seed production, certain quality traits, and the like. Accordingly efficient transformation systems for broccoli, cabbage, cauliflower, kale and other Brassica vegetables would be valuable. Many Brassica vegetable species of commercial interest do not respond well or at all to the methods previously described. A transformation method is required which is useful for substantially all of the Brassica species and especially those previously recalcitrant to transformation.
In addition to obstacles encountered within the genus Brassica, other plant genera have exhibited similar phenomena. Many commercially important plant species or genera are difficult to transform and typically only a narrow range of specific genotype is amenable to transformation. This includes crops such as cotton, soybean, sunflower, cereal crops such as wheat, barley, rice and corn as well as many ornamental and vegetable crops. Often, a wide range of genotypes are amenable to culture and regeneration, but the combination of the selection and transformation process eliminates efficient regeneration and hence leads to extremely inefficient transformation of elite lines. Thus most transformation processes are conducted with a narrow germplasm, followed by characterization of the line and extensive backcrossing. Many transgenic crop varieties produced to date have been made by transformation of a specific variety that has been demonstrated as being amenable to transformation followed by extensive crossing to those varieties most suitable for widespread cultivation. This trait introgression is time-consuming and expensive.
Accordingly, methods that permit the efficient transformation of a wide range of crop species and individual varieties, are essentially genotype independent and capable of being used to impart a variety of novel traits will be of significant benefit to the industry. Furthermore, methods that avoid, as a primary step in the process, the use of a toxic selective agent which kills non-transformed cells, may provide opportunities to achieve the efficient regeneration of plants in species where this has been found to be difficult or in those plant varieties where this is currently not possible.
It has been noted that one of the most efficient transformation processes is the natural infectivity process of Agrobacterium species. Agrobacterium is a free-living Gram-negative soil bacterium. Virulent strains of this bacterium are able to infect plant tissue and induce the production of a neoplastic growth commonly referred to as a crown gall. Virulent strains of Agrobacterium contain a large plasmid DNA known as a Ti-plasmid that contains genes required for plasmid transfer and replication as well as a region of DNA that is called T-DNA. The T-DNA region is bordered by T-DNA border sequences that are crucial to the DNA transfer process. These T-DNA border sequences are recognized by the vir genes encoded on the Ti-plasmid and the vir genes are responsible for the DNA transfer process. Thus the native Ti-Plasmid contains the vir genes as well as the T-DNA region and the T-DNA borders required for efficient DNA transfer to a recipient plant cell. The entire complement of these genetic elements, in conjunction with genes encoded on the bacterial chromosome allow the efficient transfer of the T-DNA region into plant cells with subsequent stable integration and expression of the various genes encoded by the T-DNA.
The T-DNA transferred and integrated into the plant cell nucleus contains a number of genes that encode enzymes for the production of unusual amino acids (opines) and genes that encode enzymes capable of producing plant hormones and genes responsible for the modulation of plant development. As such, there are a relatively large number of genes within the T-DNA that are transferred to the plant cell, the function of all of genes has not been completely elucidated in the Agrobacterium genus. However, the primary effect of the genes in the T-DNA are at the level of plant cell growth and development. Following infection by Agrobacterium, the plant cells infected with the T-DNA undergo an uncontrolled proliferation due to the activity of the T-DNA genes. These genes, referred to as “oncogenes” because of the phenotype they confer, permit hormone-independent growth of transformed cells in culture. The expression of these various oncogenes causes the formation of gall callus, callus that is not capable of regeneration to differentiated complex plant structures such as fertile shoots, or whole plants.
The process of gall or tumor formation is very efficient, essentially 100% of inoculated Agrobacterium susceptible plant tissue will form a tumor. Thus the natural Agrobacterium DNA transfer process is exceedingly efficient. In addition, the physiological processes of crown gall formation are also very efficient in conferring a transformed phenotype. Furthermore, most wild-type Agrobacterium have broad host range and are capable of transferring large segments of DNA, typically T-DNA transferred to plant cells contains up to 25 kilobases of DNA (e.g. nopaline strains) or more (e.g. octopine strains). Thus the naturally occurring DNA transfer system of Agrobacterium provides an efficient means to transfer DNA into a plant cell and the subsequent formation and identification of transformed tissue.
Accordingly, numerous methods of using Agrobacterium to transfer DNA into plant cells have been developed. Typically these have been based on the engineering of the Ti-plasmid to no longer contain the genes responsible for altered morphology (“oncogenes”) and replacing these genes with a recombinant gene encoding a trait of interest. Linked to this trait of interest is a gene encoding a selectable marker such that cells that receive the DNA can be selected. Usually the vir genes are left intact, however some methods include the alteration of the vir genes in one form or another.
There are two primary types of Agrobacterium based plant transformation systems, binary and co-integrate methods. The use of the binary transformation system is described by Schilperoort et al in U.S. Pat. No. 4,940,838. An example of the co-integrate system is found in Fraley et al., Biotechnology, 3:629–635, 1985. Both the co-integrate and binary systems are based on replacing the normally oncogenic complement of Agrobacterium genes with engineered, non-oncogenic DNA, typically DNA that comprises a selectable marker and a gene encoding a novel trait. The T-DNA border repeats are maintained in both systems and the natural DNA transfer process is used to transfer the portion of DNA located between the T-DNA borders into the plant cell. Thus, these transformation methods avoid the problems associated with recovery of morphologically normal plant cells when using methods wherein the oncogenic region of the Ti-plasmid is inserted into a plant cell. Therefore, these methods rely on the presence of a selectable marker to recovery transformed cells since the selection based on the activity of the oncogenes can no longer be used.
It has been recognized that the presence of oncogenes within the DNA transferred to plant cells prevents the regeneration of morphologically normal plants. It has also been recognized that each of the various oncogenes encoded by the T-DNA has a different role and the total activity of the various oncogenes leads to crown gall formation. Independently each of the primary oncogenes has a defined function and typically the presence of only one or two functional oncogenes leads to the formation of morphologically abnormal plants. This is in contrast to the crown galls produced by the activity of all of the oncogenes, crown galls (or callus derived from crown gall tissue) cannot regenerate or form morphologically normal plants. Indeed, crown gall tissue is not capable of regeneration to differentiated plant tissue. Accordingly crown gall tissue has been considered to represent a terminal tissue in the Agrobacterium infectious process. Thus it has been recognized in the art that the full complement of oncogenes contained within the T-DNA eliminates the potential for recovery of morphologically normal plants.
However, it has been shown in the art that one or two oncogenes can be included in a transformation vector for plants and the activity of these oncogenes used as a method to screen for transformed cells. For example, Ebunuma et al., (Proc. Natl. Acad. Sci. USA 94:2117–2121, 1997, U.S. Pat. No. 5,965,791) describe a plant transformation vector that comprises the isopentyl transferase gene from Agrobacterium (typically referred to as oncogene 4) as a means to identify transformed plant cells. In this method, the activity of the oncogene causes the production of cytokinin, a plant hormone that causes shoot production. In this case, a “shooty” mutant is produced (a morphologically abnormal shoot) and the plant cells are selected on the basis of abnormal shoot formation as well as selection for resistance to kanamycin or herbicides. As an added feature of the method, the oncogene is bordered by transposition elements that permit the eventual loss of the oncogene from the transformed plant cell. The arrangement of DNA in the vector is such that the gene of interest remains in the transformed plant cell.
Although this method relies on the activity of oncogene 4 to discriminate transformed versus non-transformed cells, the method requires visual discrimination of regenerating plant shoots, can lead to the formation of chimaeric plants and requires various tissue culture manipulations to recover single cell derived transgenic plants.
Indeed the method described in U.S. Pat. No. 5,965,791 relies on the regeneration of morphologically abnormal plant structures as a marker for transformation. The morphological abnormality induction gene causes the formation of abnormal plant structures, and in many cases somataclonal and culture variation lead to the same phenotypic effect, thus further analysis is required to fully obtain transgenic plants. Thus the method has limitations and may not provide a convenient means to transform recalcitrant plant species or varieties. The method still relies on the use of a dis-armed Agrobacterium strain and the efficiency of transformation is correspondingly reduced relative to the natural formation of crown galls.
Similarly, it has been shown that the oncogene 2 can be used for discrimination of transformed plant cells, and recovery of transgenic plants following regeneration and selection of transgenic cells on appropriate media or under appropriate selective conditions. Keller et al., (International Publication No. WO 00/37060) demonstrates that the isolated oncogene 2 can be used for discrimination of transgenic plant cells following transformation and culturing on selective media. Abnormal plantlets are produced in the presence of the oncogene 2 enzyme and an appropriate substrate for the enzyme. As above, the selection of a transformed plant relies on the formation of a morphologically abnormal structure.
In both of these examples, the usual transfer process of DNA from Agrobacterium to a plant cell occurs as a result of the modification of a plant transformation vector to comprise a binary vector system and a dis-armed T-DNA region. These modifications have typically included the elimination of most if not all of the natural sequences found within the T-DNA, including many open reading frames and genes whose function is not well understood but likely to play an important role in the efficiency of the transfer and integration process. These oncogenes utilized are modified and outside of the normal environment that characterizes their naturally occurring roles in the formation of crown gall tissue.
However, the natural T-DNA transfer process and subsequent formation of crown galls or plant tumors with wild-type T-DNA is a highly efficient process. The process is usually very efficient even on plant varieties and species that are recalcitrant to usual tissue-culture protocols for the introduction of DNA by Agrobacterium and recovery of transgenic plants (for example various Brassica species or cotton). In most instances crown galls will form easily on decapitated plants or wounded stems, even on plant genotypes recalcitrant to transformation using typical dis-armed vectors. Crown gall formation is also easily scored and true galls (i.e., those carrying the entire T-DNA region) are not capable of forming any differentiated plant tissue. Although spontaneous shoots or roots can form with some Agrobacterium strains, usually crown galls do not form differentiated cells, even morphologically abnormal differentiated cells unless one or more of the encoded oncogenes undergoes a mutation or loss of function. Crown galls can also be easily cultured by simple growth on hormone free minimal media. Crown galls can also be made bacteria free by culturing in antibiotic solution for a period of time. Thus it is a trivial process to obtain bacteria-free, transformed plant cells from many different plant species comprising intact oncogenic T-DNA.
Thus, a method for plant transformation that could take advantage of this efficient process, along with the easy means of identifying the transformed cells (by hormone-independent growth in culture) can provide many advantages including efficiency of transformation and selection of transformed plant cells. However, the art teaches that when the naturally occurring oncogenic region is used, recovery of morphologically normal plant cells is not possible. Thus, methods for recovering morphologically normal plant cells following the formation of crown galls are not available and hence the efficiency and wide host range of the natural T-DNA transfer process cannot be used as a means to produce morphologically normal transformed plants from a wide range of plant species.